Method for using tropoelastin and for producing tropoelastin biomaterials

ABSTRACT

It is a general object of the invention to provide a method of effecting repair or replacement or supporting a section of a body tissue using tropoelastin, preferably crosslinked tropoelastin and specifically to provide a tropoelastin biomaterial suitable for use as a stent, for example, a vascular stent, or as conduit replacement, as an artery, vein or a ureter replacement. The tropoelastin biomaterial itself can also be used as a stent or conduit covering or coating or lining.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent Ser. No. 08/797,770filed Feb. 7, 1997 (pending) which is a continuation-in-part of U.S.Pat. No. 5,989,244 issued Nov. 23, 1999 and a continuation-in-part ofU.S. Pat. No. 5,990,379 issued Nov. 23, 1999.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Grant No. DAMD17-96-1-6006 awarded by U.S. Army Medical Research Acquisition Activity.The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to a method for using tropoelastin, and moreparticularly to a method for producing tropoelastin biomaterials.

Elastic fibers are responsible for the elastic properties of severaltissues such as skin and lung, as well as arteries, and are composed oftwo morphologically distinct components, elastin and microfibrils.Microfibrils make up the quantitatively smaller component of the fibersand play an important role in elastic fiber structure and assembly.

The most abundant component of elastic fibers is elastin. The entropy ofrelaxation of elastin is responsible for the rubber-like elasticity ofelastic fibers. In vertebrates elastin is formed through the secretionand crosslinking of tropoelastin, the 72-kDa biosynthetic precursor toelastin. This is discussed, for example, in an article entitled“Oxidation, Cross-linking, and Insolubilization of RecombinantCrosslinked Tropoelastin by Purified Lysyl Oxidase” by Bedell-Hogan, etal. in the Journal of Biological Chemistry, Vol. 268, No. 14, on pages10345-10350 (1993).

In vascular replacement and repair, the best current option is toimplant autologous veins and arteries where the obvious limit is thesupply of vessels which can be sacrificed from the tissues they wereintended to service. Autologous vein replacements for damaged arteriesalso tend to be only a temporary measure since they can deteriorate in afew years in high pressure arterial circulation.

When autologous graft material is not available, the surgeon must choosebetween sacrificing the vessel, and potentially the tissue itsub-served, or replacing the vessel with synthetic materials such asDacron or Gore-tex. Intravascular compatibility indicate that several“biocompatible polymers”, including Dacron, invoke hyperplasticresponse, with inflammation particularly at the interface between nativetissue and the synthetic implant. Incomplete healing is also due, inpart, to a compliance mismatch between currently used syntheticbiomaterials and native tissues.

Thirty to forty percent of atherosclerotic stenoses that are opened withballoon angioplasty restenose as a result of ingrowth of medial cells.Smooth muscle ingrowth into the intima appears to be more prevalent insections of the artery where the internal elastic lamina (IEL) of theartery is ripped, torn, or missing, as in severe dilatation injury fromballoon angioplasty, vessel anastomoses, or other vessel trauma thatresults in tearing or removal of the elastic lamina.

Prosthetic devices, such as vascular stents, have been used with somesuccess to overcome the problems of restenosis or re-narrowing of thevessel wall resulting from ingrowth of muscle cells following injury.However, metal stents or scaffolds being deployed presently innon-surgical catheter based systems to scaffold damaged arteries areinherently thrombogenic and their deployment can result in catastrophicthrombotic closure. Metal stents have also been well demonstrated toinduce a significant intimal hyperplastic response within weeks whichcan result in restenosis or closure of the lumen. Optimal arterialreconstruction would restore the arterial architecture such that normalvascular physiology and biology would be re-established thus minimizingacute and long-term maladaptive mechanisms of vascular homeostasis.Until relatively recently, the primary methods available for securing aprosthetic material to tissue (or tissue to tissue) involved the use ofsutures or staples. Fibrin glue, a fibrinogen polymer polymerized withthrombin, has also been used (primarily in Europe) as a tissue sealantand hemostatic agent.

Damage to the arterial wall through disease or injury can involve theendothelium, internal elastic lamina, medial smooth muscle andadventitia. In most cases, the endogenous host response can repair andreplace the endothelium, the smooth muscle and the adventitial layersover a period of weeks to months depending upon the severity of thedamage. However, elastin does not undergo extensive post-developmentalremodelling and the capacity for elastin synthesis declines with age.(See “Regulation of Elastin Synthesis in Organ and Cell Culture” byJeffrey M. Davidson and Gregory C. Sephel in Methods in Enzymology 144(1987) 214-232.) Therefore, once damaged, elastic fibers are notsubstantially reformed. Neosynthesis of elastin in arterial wallssubject to hypertension or neointimal hyperplasia represents the mostsignificant example of post developmental elastin synthesis. Thissynthesis results in elastic structures mostly composed of elastinfibrils whose organization is unlike normal elastin architecture andprobably contributes little to the restoration of normal vascularphysiology.

In animal models of intimal hyperplasia or atherosclerosis it is wellaccepted that disruption of the internal elastic lamina is aprerequisite to reliable production of intimal hyperplasia oratherogenesis in large animals or primates. (See Schwartz R. S., et al.,in an article entitled “Restenosis After Balloon Angioplasty: PracticalProliferation Model In Porcine Coronary Arteries” in Circulation 1990:82: 2190-2200.) This observation is supported by several lines ofevidence that suggest a role for elastin in the biological regulation ofseveral cell types. Pathological studies indicate that elastin providesa secure attachment for endothelial cells and can act as a barrier tomacromolecules such as mitogens and growth factors preventing thesemolecules from entering the media of blood vessels. Lipids, foamymacrophages, and other inflammatory cells do not appear to enter theintima as readily when a substantial and continuous elastin membrane ispresent immediately to the endothelium according to Sims, F. H., et al.,in an article entitled “The Importance of a Substantial Elastic LaminaSubjacent to the Endothelium in Limiting the Progression ofAtherosclerotic Changes” in Histopathology (1993) at 23:307-317. Inaddition, it has been shown by Ooyama, Toshiro and Sakamoto thatchemotactic effects of soluble elastin peptides and platelet derivedgrowth factor are inhibited by substratum bound elastin peptides. (See“Elastase in the Prevention of Arterial Aging and the Treatment ofAtherosclerosis.) (See “The Molecular Biology and Pathology of ElasticTissues” edited by Chadwick, Derek J. and Jamie A. Goode, John Wiley andSons Ltd., Chichester, England (1995).) In vitro experiments show thatalpha elastin suppresses the phenotypic transition (contractile tosynthetic) of rabbit arterial SMC by interacting with a 130 kDa cellsurface elastin binding protein for cell binding sequence VGVAPG. Rabbitsmooth muscle cells adhering to elastic fibers appears to favor thecontractile over the synthetic state which is identified with restonoticresponses to injury. (See “Changes in Elastin Binding Proteins DuringPhenotypic Transition of Rabbit Arterial Smooth Muscle Cells in PrimaryCulture” by Yamamoto, et al. in Experimental Cell Research 218 (1995)pp. 339-345.) Similar work by Ooyama and colleagues has demonstratedthat the phenotypic change of smooth muscle cells from the contractileto the modified type is significantly retarded when the cells are grownon elastin coated dishes.

Until relatively recently, the primary methods available for securing aprosthetic material to tissue (or tissue to tissue) involved the use ofsutures or staples. Fibrin glue, a fibrin polymer polymerized withthrombin, has also been used (primarily in Europe) as a tissue sealantand hemostatic agent.

Laser energy has been shown to be effective in tissue welding arterialincisions, which is thought to occur through thermal melting of fibrin,collagen and other proteins. The use of photosensitizing dyes enhancesthe selective delivery of the laser energy to the target site andpermits the use of lower power laser systems, both of which factorsreduce the extent of undesirable thermal trauma.

The present invention combines the advantages of tropoelastin-basedproducts with the advantages of laser welding techniques, and provides aunique method of tissue repair and replacement. The invention makespossible tissue prostheses (particularly, vascular prostheses) that areessentially free of problems associated with prostheses known in theart.

Arterial replacement or reconstruction using tropoelastin basedbiomaterials not only may provide normal strength and elasticity butalso may encourage normal endothelial re-growth, inhibit smooth musclecell migration and thus restore normal vascular homeostasis to a degreenot currently possible with synthetic grafts.

U.S. Pat. No. 4,589,882 is directed to a method for producing syntheticelastomeric polypeptide biomaterial which replicates a portion of thecrosslinked tropoelastin polypeptide sequence. This syntheticelastomeric polypeptide biomaterial can be employed in repairing anatural elastic system of an animal body.

U.S. Pat. Nos. 4,721,096 and 4,963,489, which are incorporated herein byreference, disclose a three-dimensional cell culture system in which aliving stromal tissue is prepared in vitro by a framework composed of abiocompatible, non-living material formed into a three dimensionalstructure having interstitial spaces. Collagen has been considered for abiodegradable biomaterials for use as a framework for athree-dimensional, multi-layer cell culture system (see U.S. Pat. No.4,721,096 and No. 4,963,489).

An improved three-dimensional cell culture systems in which metaboliccycling optimizes the formation of extracellular matrix by cells grownon a three dimensional matrix is disclosed in U.S. Pat. No. 5,478,739which is herein included as a reference. U.S. Pat. No. 5,478,739 reportsproduction of collagens I, III, and IV, fibronectin, decorin, andnon-sulfated glycosaminoglycans by cells in a three dimensional culture.

SUMMARY OF THE INVENTION

It is a general object of the invention to provide a method of effectingrepair or replacement or supporting a section of a body tissue usingtropoelastin, preferably crosslinked tropoelastin.

It is a specific object of the invention to provide a tropoelastinbiomaterial suitable for use as a stent, for example, a vascular stent,or as conduit replacement, for example, as an artery, vein or a ureterreplacement. The tropoelastin biomaterial itself can also be used as astent or conduit covering or coating or lining.

It is a further object of the invention to provide a tropoelastin graftmaterial suitable for use in repairing a lumen wall.

It is another object of the invention to provide a tropoelastin materialsuitable for use in tissue replacement or repair in, for example,interior bladder replacement or repair, intestine, tube replacement orrepair such as fallopian tubes, esophagus such as for esophagealvaricies, ureter, artery such as for aneurysm, vein, stomach, lung,heart such as congenital cardiac repair, or colon repair or replacement,or skin repair or replacement, or as a cosmetic implantation or breastimplant.

It is also an object of the invention to provide a method of securing atropoelastin biomaterial to an existing tissue with or without the useof sutures or staples.

The subject invention relates to method for using a tropoelastin polymerand for producing a tropoelastin biomaterial. Such methods compriseproviding a tropoelastin monomer and then polymerizing same ashereinafter described. This will form a tropoelastin polymer which canbe formed into a biocompatible tropoelastin biomaterial from saidtropoelastin polymer for use in biomedical applications. For example, asshown in FIG. 1, the tropoelastin mononer can be formed into afilamentous structure by coacervation using moderating heating to effectsame. Then, the filamentous tropoelastin is crosslinked using acrosslinking agent, such as lysyl oxidase, to form a crosslinkedfilamentous tropoelastin material. Finally, the tropoelastin polymer isformed into a layer of biocompatible tropoelastin biomaterial. It isthis biocompatible tropoelastin biomaterial which can be used in thehereinafter described biomedical applications.

The subject invention provides a biocompatible, tropoelastin biomaterialformed into a three-dimensional structure. This structure can be used,for example, in a stromal support matrix populated with actively growingstromal cells. The stromal support matrix, which are preferablyfibroblasts, can then be used to provide support, growth factors, andregulatory factors needed to sustain long-term active proliferation ofcells in culture. A living stromal tissue can be prepared comprisingstromal cells and connective tissue proteins naturally secreted by thestromal cells which are attached to and substantially enveloping aframework composed of a biocompatible, non-living material formed intothree dimensional structure having interstitial spaces bridged by thestromal cells. The stromal cell systems contemplated herein aredescribed in the following U.S. patents of Advanced Tissue Sciences,Inc. (formerly Marrow-Tech Incorporated), which are incorporated hereinby reference:

U.S. Pat. No. 5,478,739, U.S. Pat. No. 5,460,939, U.S. Pat. No.5,443,550, U.S. Pat. No. 5,266,480,

U.S. Pat. No. 5,518,915, U.S. Pat. No. 5,516,681, U.S. Pat. No.4,963,489, U.S. Pat. No. 5,032,508, U.S. Pat. No. 4,721,096,

U.S. Pat. No. 5,516,680, U.S. Pat. No. 5,512,475, U.S. Pat. No.5,510,254, and U.S. Pat. No. 5,160,490.

The tropoelastin structure can also have a cellular lining of humancells. The cells can be derived autologously, or otherwise, and formedinto a lining on one of the major surfaces of the tropoelastin layer.Preferably, the cells which are employed to form such a lining are oneendothelial cells and/or epithelial cells and/or urothelial cells.

The tropoelastin structure can also be formed into a biocompatiblelining for mechanical structures to ensure their continued internal usein a human body. Examples of this are biocompatible linings for heartvalves, heart implants, dialysis equipment, or oxygenator tubing forheart-lung by-pass systems.

The subject invention is directed to a method for producing atropoelastin biomaterial, typically a crosslinked tropoelastin material,which is fused onto a tissue substrate, and the tropoelastin biomaterialitself. It is also directed to a method for using that tropoelastinbiomaterial, a method for producing a tropoelastin biomaterial fusedonto a tissue substrate, and a prosthetic device and a method ofproducing a prosthetic device including tropoelastin,

The present invention also relates to a method of repairing, replacingor supporting a section of a body tissue using tropoelastin. The methodcomprises positioning tropoelastin at the site of the section andbonding the biomaterial to the site or to the tissue surrounding thesite. The bonding is effected by contacting the biomaterial and thesite, or tissue surrounding the site, at the point at which said bondingis to be effected, with an energy absorbing agent. The agent is thenexposed to an amount of energy absorbable by the agent sufficient tobond the biomaterial to the site or to the tissue surrounding the site.

A tissue-fusible tropoelastin biomaterial can be produced using themethod of the present invention which comprises a layer of tropoelastinbiomaterial and a tissue substrate each having first and second outersurfaces, and an energy absorbing material applied to at least one ofthe outer surfaces. Preferably, the energy absorbing material penetratesinto the biomaterial.

The energy absorbing material is energy absorptive within apredetermined range of light wavelengths depending on materialthickness. The energy absorbing material is chosen so that when it isirradiated with light energy in the predetermined wavelength range, theintensity of that light will be sufficient to fuse together one of thefirst and second outer surfaces of the tropoelastin biomaterial and thetissue substrate. Preferably, the first and second outer surfaces of thetropoelastin biomaterial are major surfaces.

Typically, an energy absorbing material is indirectly irradiated bydirecting the light energy first through the tropoelastin biomaterial ortissue substrate and then to the energy absorbing material. Although theenergy absorbing material can be applied directly to the tissuesubstrate, it is not the preferred method because of the difficulty incontrolling penetration into the intertices of the tissue substrate.

In a preferred method of this invention, the energy absorbing materialcomprises a biocompatible chromophore, more preferably an energyabsorbing dye. In one form of the present invention, the energyabsorbing material is substantially dissipated when the tropoelastinbiomaterial and the tissue substrate are fused together. In another formof this invention, the energy absorbing material comprises a materialfor staining the first or second surface of the tropoelastinbiomaterial. The energy absorbing material can also be applied to one ofthe outer surfaces of the biomaterial by doping a separate elastin layerwith an energy absorbing material and then fusing the doped separateelastin layer to the tropoelastin biomaterial. In any case, the energyabsorbing layer is preferably substantially uniformly applied to atleast one of the outer surfaces, typically in a manner wherein theenergy absorbing material substantially covers the entire outer surfaceof the tropoelastin biomaterial.

Some of the key properties which effect the method of the presentinvention regarding fusing the tropoelastin biomaterial and tissuesubstrate include the magnitude of the wavelength, energy level,absorption, and light intensity during irradiation with light energy ofthe energy absorbing material, and the concentration of the energyabsorbing material. These properties are arranged so that thetemperature during irradiation with light energy for period of timewhich will cause fusing together of one of the first and second outersurfaces of the tropoelastin biomaterial and the tissue substrate isfrom about 40 to 140 degrees C., and more preferably from about 50 to100 degrees C., but if well localized to the biomaterial tissueinterface, can be as high as 600 degrees C. Furthermore, the averagethickness of the energy absorbing material in the preferred method ofthis invention is from about 0.5 to 300 microns.

The subject invention is also directed to a prosthetic device comprisinga support member comprising a stent, a conduit or a scaffold having alayer of tropoelastin material located on the support member. In thepreferred case, the layer of the tropoelastin biomaterial completelysurrounds the support member.

The support member of the prosthetic device is preferably formed of ametal or a synthetic material. The metal preferably comprises titanium,tantalum, stainless steel or nitinol. The synthetic material typicallycomprises a polymeric material. This polymeric material is generallyselected from a group consisting of polyethylene terepthalate (Dacron),Gore-tex, teflon, polyolefin copolymer, polyurethane and polyvinylalcohol. The support member can be formed from a hybrid polymercomprising a synthetic polymeric material and a natural polymericmaterial including fibrin and/or elastin. The support member can also beformed from a biological material, preferably from collagen.

The prosthetic device can comprise a layer of tropoelastin biomaterial.Preferably this layer comprises a covering, a coating, or a lining forthe support member. The tropoelastin biomaterial can be formed bypolymerization, or formed to a suitable size and shape by molding. Thepolymerized tropoelastin biomaterial can also be further cross-linkedusing gamma radiation and/or a cross-linking agent. In one form of theinvention, the tropoelastin biomaterial is formed into a sheet, and thesheet is employed as the covering for the support. The sheet can also beattached to the support by grafting, by mechanical bonding, or by laserbonding.

The prosthetic device of this invention is implantable within an artery,a vein, an esophagus, an intestine, a colon, a ureter, a liver, aurethra, or a fallopian tube.

A drug can be incorporated into the layer of tropoelastin materialthereby decreasing the need for systemic intravenous or oralmedications. Also, photodynamic therapy drugs (“PTD”) which areactivated with light can be employed herein.

In use, a method for producing the prosthetic device of the presentinvention comprises first providing a layer of tropoelastin biomaterialand a support member comprising a stent, a conduit or a scaffold. Then,the layer of tropoelastin biomaterial is applied to the support memberto form the prosthetic device. For example, a layer of tropoelastinmaterial can be located on the support member and can be fused together.This can be accomplished by applying an energy absorbing material, whichis energy absorptive within a predetermined range of light wavelengths,to the tropoelastin biomaterial in an amount which will cause fusingtogether thereof. Thus, the energy absorbing material is irradiated withlight energy in the predetermined wavelength range with an intensitysufficient to fuse together the tropoelastin biomaterial on said supportmember thereby fusing together the tropoelastin biomaterial on thetissue substrate.

Further objects and advantages of the invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a reaction of tropoelastinmonomers for producing crosslinked tropoelastin.

FIG. 2 is a schematic representation of a reaction of tropoelastinmonomers in the presence of fibroblasts for producing crosslinkedfibroblast-tropoelastin matrix.

FIG. 3 is a schematic representation of a reaction of tropoelastinmonomers in the presence of a preformed collagen lattice for producingtropoelastin fibrils supported on the collagen latice structure.

DETAILED DESCRIPTION Monomer Synthesis

Tropoelastin monomer is the soluble biosynthetic precursor to elastin.It is formed naturally in vetebrates. Tropoelastin can be isolated fromthe aortas of copper deficient swine by known methods such as describedby E. B. Smith, Atherosclerosis 37 (1980) tropoelastin is a 72-kDapolypeptide which is rich in glycine, proline, and hydrophobic aminoacids. The exact amino acid composition of tropoelastin differs fromspecies to species. Any polypeptide moiety that has art-recognizedhomology to tropoelastin can be considered a tropoelastin monomer forthe invention.

The tropoelastin can be isolated from mammalian tissue or produced usingrecombinant expression systems. Furthermore, tropoelastin splicevariants from any species can also be used for the invention.

The following are exemplary descriptions of methods of producingtropoelastin monomers used in the invention:

1. Tropoelastin can be extracted from mammals which have been placed oncopper deficient or lathyritic diets. The deficiency of copper in themammalian diet inhibits lysyl oxidase resulting in the accumulation oftropoelastin in elastin rich tissues. Copper deficient animals are grownrapidly on a diet composed largely of milk products and must be keptisolated from contaminating sources of copper. The protocol for raisingcopper deficient swine is detailed by L. B. Sandberg and T. B. Wolt.Production of Soluble Elastin from Copper Deficient Swine. Methods inEnzymology 82 (1982) 657-665. 150 mg of tropoelastin can be extractedfrom a 15-kg copper-deficient swine.

2. In a method similar to copper deficiency method in No. 1 above,feeding animals chemicals that effectively inhibit the action of lysyloxidase (lathyrogens) also restricts the conversion of tropoelastin toamorphous elastin. This method produces similar yields of tropoelastinto copper-deficient swine. However, the special cages, water and dietrequired to raise copper-deficient animals are not required herein. Toinduce lathyrisim, animal diets are supplemented with 0.1% by weighta-aminoacetonitrile-HCl and 0.05% a-aminocaproic acid as described byCeleste B. Rich and Judith Ann Foster, Isolation of SolubleElastin-Lathyrism. Methods in Enzymology 82 (1982) 665-673.

3. Tropoelastin can also be produced by mammalian cell culture systems.Short term cultivation of bovine vascular endothelial cells, nuchalligament fibroblasts from cows and sheep, human skin fibro-blasts, andvascular smooth muscle cells from pigs and rabbits results in theaccumulation of tropoelastin in the culture medium.

4. Recombinant tropoelastin produced by a protein expression system isthe preferred monomer for the invention. Recombinant protein technologyis the transfer of recombinant genes into host organisms that grow andconvert nutrients and metabolites into recombinant protein products.Using this technology, cDNA encoding tropoelastin can be cloned andexpressed in protein expression systems to produce biologically activerecombinant tropoelastin. Functionally distinct hydrophobic domains andlysine rich crosslinking domains are encoded in separate exons. Thisexistence of multiple splice variants of tropoelastin in several speciescan be attributed to Cassette-like alternative splicing of elastinpre-mRNA. Expression of different recombinant splice variants oftropoelastin can produce proteins with distinct qualities. In addition,site directed in vitro mutagenesis can be used to alter the polypeptidesequence of the naturally occurring gene, thus creating alternatepolypeptides with improved biological activity and physical properties.Expression of the full length elastin cDNA clone, cHEL2 and subsequentpurification of recombinant human tropoelastin (rTE) has been achievedby Joel Rosenbloom, William R. Abrams, and Robert Mecham. ExtracellularMatrix 4: The Elastic Fiber. The Faseb Journal 7 (1993) 1208-1218. rTEproduced by the methods of Rosenbloom et al. can be used for theinvention, however, the methods are not considered to be part of thepresent invention. In addition, the invention is not limited to rTEproduced from the expression of cHEL2. rTE produced from the expressionof any tropoelastin genomic or cDNA can be used for the invention.

To help overcome the moderate yields of rTE recovered by Rosenbloom andcolleagues, Martin, Vrhovski and Weiss successfully synthesized andexpressed a gene encoding human tropoelastin in E. Coli. In constructingthe gene they tailored the rare codon bias of the synthetic sequence tomatch the known preferences of E. Coli. rTEtropoelastin produced byexpression of synthetic genes can be used for the invention.

rTE is used in the invention can be produced in non-bacterial expressionvector systems. Yeast expression vector systems are well suited forexpressing eukaryotic proteins and tropoelastin is a potentiallyexcellent candidate for expression in yeast.

For large scale heterologous gene expression, the baculovirus expressionvector system (BEVS) is particularly advantageous. BEVS has severaladvantages over other expression systems for mammalian gene expression.It is safer, easier to scale up, more accurate, produces higherexpression levels, and is ideal for suspension cultures permitting theuse of large-scale bioreactors. Generation of a recombinant baculovirusparticle carrying a clone of elastin cDNA coding for an isoform oftropoelastin is achieved through homologous recombination or sitespecific transposition and is followed by recombinant baculovirusinfection of insect cells (Sf9 or High Five) and subsequent recombinantgene expression as follows:

Elastin cDNA encoding tropoelastin is identified and isolated from acDNA library. The gene is cloned into a pFastBac or pFastBac HT donorplasmid using standard restriction endonucleases and DNA ligase. Correctinsertion of gene is verified by restriction endonuclease digestion andPCR analysis. The DNA is then transformed into DH10Bac cells whichharbor a bacmid a mini-attTn7 target site and a helper plasmid. Oncecloned into the DH10Bac cells, the elastin gene undergoes site-specifictransposition into the Bacmid. Transposition results in the disruptionof a LacZalpha gene and colonies containing recombinant bacmids arewhite. High molecular weight mini-prep DNA is prepared from selected E.Coli clones containing the recombinant bacmid and is used to transfectSF9 or High Five insect cells using CellFECTIN reagent. The insect cellsproduce actual baculovirus particles harboring the tropoelastin encodinggene. The virus particles are harvested and are subsequently used toinfect insect cells which produce high yields of the recombinant proteinproduct, tropoelastin.

Tropoelastin accumulated in elastin rich tissues by the inhibition oflysyl oxidase through copper deficiency or lathyrism can be isolated byexploiting tropoelastin's high solubility in short-chain alcohols.Modified methods of this alcohol extraction procedure can be used topurify rTE from expression hosts such as bacteria, yeast, insect, andmammalian cells in culture. Methods have been described in detail whichinvolve precipitation of tropoelastin with n-propanol and n-butanol.Tropoelastin expressed in insect cells using the pFastBac HT baculovirusexpression system (Life Technologies, Gaithersburg, Md.) can be purifiedin a single affinity chromatography step with Ni-NTA resin. Theinvention is not limited to any particular method of tropoelastinisolation or purification.

Polymer Synthesis

In tissue, tropoelastin is naturally crosslinked by several tetra andbifunctional cross-links to form elastin. These crosslinks arise throughthe oxidative deamination and condensation of lysyl side chains. Bothbifunctional lysinonorleucine and allysine aldol and tetrafunctionaldesmosine crosslinks are formed. Tetrafunctional desmosine crosslinksare a distinguishing feature of elastin. Tropoelastin can be convertedto a tropoelastin biomaterial by oxidative deamination of lysyl residuesand the subsequent crosslinking of the monomeric moiety catalyzed by thecopper dependent enzyme lysyl oxidase (protein-lysine 6-oxidase).

A primary purpose of the invention is to produce cross-linked elasticmatrices that are identical to or closely mimic those found naturally inelastic tissue. It is, therefore, advantageous to crosslinktropo-elastin monomers with the same bifunctional and tetrafunctionalcross-links found in elastin. However, the invention is not limited tothese naturally occurring cross-links and any type of cross-link formedbetween tropoelastin monomers, whether produced chemically,enzymatically or radiatively, can be used for the invention.

Crosslinking tropoelastin with lysyl oxidase will produce matrices thatclosely resemble or imitate naturally occurring ones. Lysyl oxidaseprotein-lysine 6-oxidase) catalyzes the oxidation of lysine residues toa peptidyl α-aminoadipic -α-semialdehyde. This aldehyde residuespontaneously condenses with neighboring aldehydes or α-amino groupsforming interchain or intrachain crosslinkages (Kagan, 1991). Lysyloxidase from any source can be used so long as the tropoelastin it isintended to oxidize is a suitable ligand. Lysyl oxidase is typicallyextracted from bovine aorta and lung, human placentas, and rat lung with4 to 6 M urea extraction buffers. Recombinantly produced lysyl oxidasecan also be used to cross-link tropoelastin. Recombinant tropoelastin(rTE26A) has been cross-linked with lysyl oxidase in 0.1 M sodiumborate, 0.15 M NaCl, pH 8.0 when incubated for 24 hr at 37° C.(Bedell-Hogan, 1993). Another preferred method of crosslinkingtropoelastin is with γ-irradiation. γ-irradiation causes formation offree radicals which can result in crosslink formation. 20 mrad ofγ-irradiation has been shown to crosslink an elastin like polypeptide,poly(GLy-Val-Gly-Val-Pro), into an elastomeric matrix and has increasedthe elasticity and strength of a elastin-fibrin biomaterial. Theaddition of chemical agents that form crosslinks when activated withirradiation can also be used. Sulfur derivatives combined withγ-irradiation been shown to further increase the strength of anelastin-fibrin biomaterial. Chemical crosslinking reagents such asglutaraldelhyde may also be used to cross-link tropoelastin matrices.

A preferred method of organizing tropoelastin monomers into fibrousstructures prior to cross-linking is by taking advantage of the propertyof coacervation exhibited by tropoelastin. Tropoelastin is soluble inwater at temperatures below 37° C., however, upon raising thetemperature to 37° C. tropoelastin aggregates into a filamentousstructure called a coacervate. Formation of tropoelastin coacervates maybe a natural step prior to cross-link formation during elastogenesis intissue. Coacervated tropelastin can be crosslinked by lysyl oxidaseunder the appropriate conditions to produce filamentous elastin fibrils.Alignment may be facilitated by exposure of the tropoelastin coacervatesto a magnetic field prior to crosslinking.

Collagen is the major structural polymer of connective tissues.Artificial collagen fibers have been produced from soluble collagen Iextracts. Fibers such as these can be formed into scaffoldings ontowhich tropoelastin can be cross-linked into amorphous insoluble elastinproducing a elastin/collagen composite (see FIG. 3). The collagen fiberslend form and tensile strength to the tropoelastin material and thecrosslinked tropoelastin fibrils lend elasticity thus creating acomposite material that very nearly approximates naturally occurringconnective tissue.

Proteoglycans are major constituents of the extracellular matrix. Theaddition of Hyaluronic acid, dermatan sulfate, keratane sulfates, orChondroitin sulfates as co-materials may further the strength andcohesion of the material. In addition, cell function is in partcontrolled by the extracellular matrix. Fibronectin, vitronectin,laminin nad collagen, as well as various glycosaminoglycans all mediatecell adhesion. Fibronectin has several roles in the connective tissuematrix. It has an organizing role in developing tissues and it plays amajor role in cell adhesion to the extracellular matrix. Incorporationof fibronectin as a co-material may improve the cell adhesion propertiesof the tropoelastin based biomaterial. Microfibrils are distributedthroughout the body, and are prevalent in elastic tissues and fibers.The presence of microfibrils during polymerization of tropoelastinmonomers may help to organize monomers yielding a material with improvedstructural organization. Also, microfibrils are known to sequestercalcium ions and are thought to play a role in protecting tropoelastinfrom chronic calcification.

Product Synthesis

The utility of tropoelastin based biomaterials may be further improvedby combining them with synthetic or natural polymer co-materials,forming composites, and by adding bioactive impregnates.

Antibiotics and/or anticoagulants or other agents can be added to thetropoelastin matrix providing localized drug therapy and preventinginfection. In surgical repair of abdominal traumatic injuries, infectionrepresents a major problem particularly when vascular prostheticimplants are used. An tropoelastin graft with antibiotic incorporationmay be ideal because it avoids sacrifice of an autologous artery or veinwhich decreases surgical time and precludes the necessity to usesynthetic prosthetic materials which may be more prone to infection thantropoelastin grafts. Bioactive impregnates may also includeanti-coagulants (Hirudin), coagulants, anti-proliferative drugs(Methatrexate), growth factors, anti-virals, and anti-neoplastics.

Small diameter vascular grafts fail at an unacceptable rate due to theirinherent throm-bogenicity. This problem may be decreased by thedeposition of a living autologous endothelial cell lining. Autologousendothelial cell transplantation can accelerate the formation of animmunologically compatible, complete endothelial lining usingmicrovascular endothelial cells derived from the adipose tissue of arecipient animal (Jarrell, et al.). In the porcine model the peritonealfat had been determined to be optimal for this purpose due to thepredominance of microvascular and endothelial cells. Followingextraction of peritoneal fat, homogenization, collagenase digestion, andcentrifugal separation, cells are expeditiously transplanted onto theluminal surface of crosslinked tropoelastin vascular grafts using anintra-operative isolation technique combined with the rapid pressuresodding techniques described by Jarrell and Williams.

The present invention constitutes a three dimensional matrix made ofelastin or tropoelastin for use as a framework for a three-dimensional,multi-layer cell culture system. Populating endogenous biologicmaterials such as a tropoelastin matrix with stromal cells is preferableto populating matrices made of synthetic biocompatible, non-livingmaterials. Synthetic biodegradable biomaterials must undergo enzymecatalyzed degradation or spontaneous hydrolysis in order to avoidpermanent chronic foreign body reactions. On the contrary, elastin is anaturally occurring protein in the extracellular matrix of many tissuesand, therefore, does not illicit a foreign body reaction. Unlikecollagen, elastin undergoes very little post-developmental remodellingor breakdown and is a relatively permanent connective tissue structureduring the life of an organism. Tropoelastin biomaterials can provide arelatively permanent, natural support matrix for three dimensional cellcultures that when implanted acts as a template for reconstruction ofthe organs and tissues. In addition the longevity and integrity ofimplanted tropoelastin is regulated in response to the biological needsof the tissue rather than environmentally induced hydrolysis orenzymatic degradation of a foreign substance.

Elastin structures constituting a framework for a three-dimensional,multi-layer cell culture system will provide intact elastic structuresnot constructed by stromal cells populating synthetic matrices. In vivoelastin production is thought to only occur during development andceases during childhood (the only exceptions being hypertension andrestenosis). Elastogenesis is a complex method and formation of matureelastic structures not likely to be achieved in relatively simple invitro cell culture systems. However, it has not been reported that suchthree dimensional cell culture systems can organize elastin intocoherent fibrous matrices analogous to those found in elastic tissues. Amethod by which to produce a living tissue graft with elastic structureand function most similar to tissue which is high in elastin content isby culturing cells in three dimensional frameworks made of elastin orelastin based biomaterials. This insures the presence of biologicallyimportant elastic structures in the living tissue grafts.

A method for both organizing tropoelastin fibrils and providing asupport for fibroblast growth is by coacervating tropoelastin monomersin solution with fibroblasts. Tropoelastin monomers mixed with stromalcells (fibroblasts) in a physiologic buffer aggregate into fibers(coacervation) upon raising the temperature of the solution to 37° C. Indoing so the fibroblasts become trapped in a loose matrix of elasticfibers. The tropoelastin fibers can be crosslinked either by includinglysyl oxidase in the buffer or a temperature sensitive recombinant formof lysyl oxidase that, for example, is inactive at 20° C. and active at37° C. or by culturing the tropoelastin-fibroblast matrix in such amanner that the fibroblasts secrete natural lysyl oxidase into thecoacervate matrix. The contraction of the fibroblasts bound to thecoacervated tropoelastin monomers could preferentially align thetropoelastin fibrils prior to crosslinking.

Sterilization

The tropoelastin biomaterial of the invention is normally secured toexisting tissue. Various techniques for effecting that attachment can beused, including art-recognized techniques, including suturing, staplesand gluing. However, in some cases it is preferred that the biomaterialbe secured using a tissue welding energy source and an agent thatabsorbs energy emitted by that source. Advantageously, the energy sourceis an electromagnetic energy source, such as a laser, and the absorbingagent is a dye having an absorption peak at a wavelength correspondingto that of the laser. The tropoelastin biomaterial and the tissue to bewelded have much less absorption of light at this wavelength and theeffect therefore is confined to a zone around the dye layer. A preferredenergy source is a laser diode having a dominant wavelength at about 808nm and a preferred dye is indocyanine green (ICG), maximum absorbance795-805 nm (see WO 91/04073). Other laser/dye combinations can also beused. It is preferred that the dye be applied to that portion of thebiomaterial that is to be contacted with and secured to the existingtissue. The dye can also be applied to the surface of the structure towhich the tropoelastin biomaterial is to be welded or secured. The dyecan be applied directly to the biomaterial or the surface of thebiomaterial can first be treated or coated (e.g. primed) with acomposition that controls absorption of the dye into the biomaterial sothat the dye is kept as a discrete layer or coating. Alternatively, thedye can be bound to the troptropoelastin biomaterial so that it issecured to the surface and prevented from leeching into the material.The dye can be applied in the form of a solution or the dye can bedissolved in or suspended in a medium which then can be applied as athin sheet or film, preferably, of uniform thickness and dyeconcentration.

Tissue welding techniques employing a soldering agent can be used. Suchtechniques are known (WO 91/04073). Any proteinaceous material thatthermally denatures upon heating can be used as the soldering agent (forexample, any serum protein such as albumin, fibronectin, Von Willebrandfactor, vitronectin, or any mixture of proteins or peptides). Solderscomprising thrombin polymerized fibrinogen are preferred, except wheresuch materials would cause undesirable thrombosis or coagulation such aswithin vascular lumens. Solders are selected for their ability to impartgreater adhesive strength between the biomaterial and the tissue. Thesolder should be non-toxic and generally biocompatible.

In accordance with the present invention, the laser energy can bedirected to the target site (e.g. the dye) directly from the laser byexposure of the tissue (e.g. during a surgical procedure). In somecases, i.e., endovascular catheter-based treatments where open surgicalexposure does not occur, the laser energy is directed to the bondingsite via optical fibers. When ICG is used as the dye, targeting mediawavelengths of around 800 nm can be used. Such wavelengths are not wellabsorbed by many tissues, particularly blood and vascular tissues,therefore, there will be a negligible effect on these tissues andthermal effects will be confined to the dye layer. The biomaterial ofthe invention similarly has little optical absorbance in this waveband,as compared to the energy absorbing dye. Thus, the laser energy can passthrough either the biomaterial or the native tissue and be absorbed bythe dye layer as shown in FIG. 1. Once the surgeon has exposed thesurface or vessel where the biomaterial reinforcement or replacement isto be effected, the dye-containing surface of the biomaterial is placedin contact with the native tissue at the site and laser energy deliveredby directing the laser beam to the desired location. The absorbance ofthe dye (e.g. ICG) layer is ideally previously or concurrentlydetermined so that the optimal amount of light for optimal bonding canbe delivered. Pressure can be used to ensure adequate approximation ofthe tissue and biomaterial. With a diode laser source, the diode laseritself, or a condenser or optical fiber based optical delivery system,can be placed against the material to ensure uniform light delivery.

In cases where a new elastin lining or new-internal elastic lamina isrequired, for example, following an open surgical endarterectomy, oncethe artery has been surgically cleared of the atheroma or other lesion,the biomaterial is then put in place, dye side down. The biomaterial canbe deployed as a flat patch or as a tubular segment. A tubular segmentcan be hollow or filled with a material that supports the lumen duringplacement and that is melted with low grade heat or dissolved or removedwith a variety of means. When necessary, a small number of surgicalsutures (e.g. stay sutures) can be used to appose the edges of thevessel together or to sew the vessel. Once the biomaterial is in place,the laser energy is directed through the vessel wall or through thebiomaterial to the absorbing dye, the appropriate laser energy havingbeen previously determined based upon the measured absorbance in thebiomaterial. Alternatively, the dye can be applied at the time of thesurgery to the biomaterial or the vessel wall or both and then laserenergy delivered. In this embodiment, absorbance can be determined atthe time of the surgery to the biomaterial or the vessel wall or bothand then laser energy delivered or with a feedback device that assessesthe adequacy of the bonding or thermal effect.

In addition to the above, the biomaterial of the invention can be usedas a patch material for use in intestinal or colon repairs whichfrequently do not heal well with current techniques, particularly whenthe patient has nutritional or other problems or when the patient is inshock, such as in the case of multiple gunshot wounds or other abdominalinjuries (see FIG. 3). The use of such a patch can, for example, sealoff intestinal contents and thereby reduce the likelihood ofperitonitis. In addition, a patch can be used on a solid organ, such asthe liver or lung, when lacerations have occurred. Similarly, thebiomaterial of the invention can be used to repair or replace portionsof the urinary system, i.e., from the calyces of the kidney on down tothe urethra. The patch can also be used to seal a defect in a cardiacchamber, such as an atrial septal defect, as well as bronchial or rectalfistulas. The biomaterial can also be used as a cerebrovascular patchfor an aneurysm. The biomaterial can be sealed in place with targetedlaser fusion. For applications where direct exposure is not possible ornot desirable, a variety of catheter or endoscopic systems can beemployed to direct the laser energy to the target site bio-materials towhich the invention relates can be used in a variety of other clinicaland surgical settings to effect tissue repair graft. For delivery ofbiomaterial in the form of an intravascular stent, the biomaterial canbe pre-mounted upon a deflated balloon catheter. The balloon cathetercan be maneuvered into the desired arterial or venous location usingstandard techniques. The balloon can then be inflated, compressing thestent (tropoelastin biomaterial) against the vessel wall and then laserlight delivered through the balloon to seal the stent in place (the dyecan be present on the outside of the biomaterial). The balloon can thenbe deflated and removed leaving the stent in place. A protective sleeve(of plastic or the like) can be used to protect the stent during itspassage to the vessel and then withdrawn once the stent is in thedesired location.

The biomaterial of the invention can also be used as a biocompatiblecovering for a metal or synthetic scaffold or stent. In such cases,simple mechanical deployment can be used without the necessity for laserbonding. Laser bonding can be employed, however, depending upon specificdemands, e.g., where inadequate mechanical bonding occurs, such as instent deployment for abdominal aortic aneurysms. An alternativecatheter-based vascular stent deployment strategy employs a temporarymechanical stent with or without a balloon delivery device.

A further catheter-based vascular stent deployment strategy employs aheat deformable metal (such as nitinol or other similar type metal)scaffold or stent or coating that is incorporated into the cathetertubing beneath the stent biomaterial. The stent is maneuvered into thedesired location whereupon the deformable metal of the stent isactivated such that it apposes the stent against the vessel wall. Laserlight is then delivered via an optical fiber based system, alsoincorporated into the catheter assembly.

The tropoelastin-based biomaterial can also be used to replace portionsof diseased or damaged vascular or nonvascular tissue such as esophagus,pericardium, lung plura, etc. The biomaterial can also be used as a skinlayer replacement, for example, in burn or wound treatments. As such,the biomaterial serves as a permanent dressing that acts as ascaffolding for epithelial cell regrowth. The biomaterial can includeantibiotics, coagulants or other drugs desirable for various treatmentsthat provide high local concentrations with minimal systemic druglevels. The tropoelastin biomaterial can be deployed with a dye on thetissue side and then fused with the appropriate wavelength and laserenergy.

In addition to repair of tubular body structures, the biomaterial of thepresent invention can also be used in organ reconstruction. For example,the biomaterial can be molded or otherwise shaped as a pouch suitablefor use in bladder reconstruction. The biomaterial of the invention canalso be molded or otherwise shaped so as to be suitable for esophagealreplacement. Again, metal or synthetic mesh could also be associatedwith the implant if extra wall support is needed so as to controlpassage of food from the pharynx to the stomach. This could be used forstenosis of the esophagus, repair from acid reflux for erosiveesophagitis or, more preferably, for refurbishing damaged esophagealsegments during or following surgery or chemotherapy for esophagealcarcinoma.

For certain applications, it may be desirable to use the biomaterial ofthe invention in combination with a supporting material having strongmechanical properties. For those applications, the biomaterial can becoated on the supporting material (see foregoing stent description), forexample, using the molding techniques described herein. Suitablesupporting materials include polymers, such as woven polyethyleneterepthalate (Dacron), teflon, polyolefin copolymer, polyurethanepolyvinyl alcohol or other polymer. In addition, a polymer that is ahybrid between a natural polymer, such as fibrin and elastin, and anon-natural polymer such as a polyurethane, polyacrylic acid orpolyvinyl alcohol can be used (see Giusti et al., Trends in PolymerScience 1:261 (1993). Such a hybrid material has the advantageousmechanical properties of the polymer and the desired biocompatibility ofthe tropoelastin material. Examples of other prostheses that can be madefrom synthetics (or metals coated with the tropoelastin basedbiomaterial or from the biomaterial/synthetic hybrids include cardiacvalve rings and esophageal stents.

The tropoelastin-based prostheses of the invention can be prepared so asto include a drug; that can be delivered, via the prostheses, toparticular body sites. For example, vascular stents can be produced soas to include drugs that prevent coagulation, such as heparin, orantiplatelet drugs such as hirudin, drugs to prevent smooth muscleingrowth or drugs to stimulate endothelial damaged esophageal segmentsduring or following surgery or chemotherapy for esophageal carcinoma orendothelial regrowth. Vasodilators can also be included.

Prostheses formed from the tropoelastin bio-material can also be coatedwith viable cells, cells from the recipient of the prosthetic device.Endothelial cells, preferably autologous (e.g. harvested duringliposuction), can be seeded onto the elastin bioprosthesis prior toimplantation (e.g. for vascular stent indications). Alternatively, thetropoelastin biomaterial can be used as a skin replacement or repairmedia where cultured skin cells can be placed on the biomaterial priorto implantation. Skin cells can thus be used to coat elastinbiomaterial.

All documents cited above are hereby incorporated in their entirety byreference. One skilled in the art will appreciate from a reading of thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

Having described and illustrated the principles of the invention in apreferred embodiment thereof, it should be apparent that the inventioncan be modified in arrangement and detail without departing from suchprinciples. I claim all modifications and variation coming within thespirit and scope of the following claims.

1. A method for producing a tissue substrate fusible biomaterialconsisting essentially of tropoelastin which is fusible to a tissuesubstrate comprising: providing a polymerizable monomer consistingessentially of tropoelastin; polymerizing said polymerizable monomer toform a polymer consisting essentially of tropoelastin: forming a layerof said biomaterial from said tropoelastin polymer having a first andsecond outer major surface; and applying an energy absorbing material,which is energy absorptive within a predetermined range of lightwavelengths, to a selected one of said first and second outer surfacesof the biomaterial in an amount which will cause fusing together of oneof said first and second outer surfaces of the biomaterial and one ofsaid first and second outer surfaces of said tissue substrate, saidenergy absorbing material penetrating into the interstices of saidbiomaterial; and irradiating the energy absorbing material with lightenergy in said predetermined wavelength range with an intensitysufficient to fuse together one of said first and second outer surfacesof the biomaterial and the tissue substrate.
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 11. The method of claim 1, wherein the tissue substrate is alive tissue substrate.
 12. The method of claim 1, wherein the averagethickness of the energy absorbing material which penetrates into theinterstices of the biomaterial is from about 0.5 to 300 microns. 13.(cancel)
 14. The method of claim 1, wherein the tissue substrate isselected from a group consisting of bladders, intestines, tubes,esophagus, ureters, arteries, veins, stomachs, lungs, hearts, colons,and skin.
 15. The method of claim 1, wherein said polymerizable monomerconsists essentially of non-mammalian tropoelastin.
 16. The method ofclaim 15, wherein said non-mammalian tropoelastin consists essentiallyof recombinant tropoelastin.
 17. The method of claim 15, wherein saidrecombinant tropoelastin is produced by a protein expression system. 18.The method of claim 1, which further includes the step of forming acellular lining of human cells on one of the major surfaces of saidbiomaterial layer.
 19. The method of claim 18, wherein said cells whichare employed to form said cellular lining are at least one ofendothelial cells, epithelial cells and urothelial cells.
 20. The methodof claim 1, which further includes the step of forming an inner liningconsisting essentially of tropoelastin for mechanical human structuresto ensure their continued internal use in a human body.
 21. The methodof claim 20, which further includes the step of forming said innerlining in heart valves, heart implants, dialysis equipment, oroxygenator tubing for heart-lung by-pass systems.
 22. The method ofclaim 1, which includes the step of introducing a drug into saidbiomaterial.
 23. A method for producing a prosthetic device comprising:providing a polymerizable monomer consisting essentially oftropoelastin; polymerizing said polymerizable monomer to form a polymerconsisting essentially of tropoelastin: forming a layer of saidbiomaterial from said tropoelastin polymer having a first and secondouter major surface; providing a support member comprising a stent, aconduit or a scaffold; and applying said layer of said biomaterial tosaid support member to form said prosthetic device.
 24. The method ofclaim 23, which includes the step of applying the layer of saidbiomaterial so that it surrounds said support member.
 25. The method ofclaim 23, which includes the step of forming said biomaterial bypolymerization.
 26. The method of claim 23, which includes the step ofmolding said biomaterial of a suitable size and shape.
 27. The method ofclaim 23, which includes the step of forming said biomaterial into asheet or tube, and then covering said support member with said sheet ortube.
 28. The method of claim 23, which includes the step of applyingsaid biomaterial layer to said support by grafting.
 29. The method ofclaim 23, which includes the step of applying said biomaterial layer tosaid support by mechanical bonding.
 30. The method of claim 23, whichincludes the step of applying said biomaterial layer to said support bylaser bonding.
 31. The method of claim 23, which includes the step ofincorporating a drug into said biomaterial layer thereby decreasing theneed for systemic intravenous or oral medications.
 32. The method ofclaim 23, wherein said support member comprises titanium, tantalum,stainless steel or nitinol.
 33. The method of claim 23, wherein saidpolymerizable monomer consists essentially of non-mammaliantropoelastin.
 34. The method of claim 33, wherein said non-mammaliantropoelastin consists essentially of recombinant tropoelastin.
 35. Themethod of claim 34, wherein said recombinant tropoelastin is produced bya protein expression system.
 36. A method for producing a biomaterial,which comprises: providing a polymerizable monomer consistingessentially of non-mammalian tropoelastin; polymerizing saidpolymerizable monomer to form a polymer consisting essentially ofnon-mammalian tropoelastin; and forming a biomaterial consistingessentially of non-mammalian tropoelastin from said polymer.
 37. Themethod of claim 36, wherein said non-mammalian tropoelastin consistsessentially of recombinant tropoelastin.
 38. The method of claim 37,wherein said recombinant tropoelastin is produced by a proteinexpression system.
 39. A method for producing a biomaterial, whichcomprises: providing a crosslinkable monomer consisting essentially oftropoelastin; crosslinking said crosslinkable monomer to form acrosslinked polymer consisting essentially of crosslinked tropoelastin;and forming a biomaterial from said crosslinked polymer consistingessentially of crosslinked tropoelastin.
 40. The method of claim 39,wherein said crosslinked tropoelastin consists essentially oftropoelastin having non-naturally occurring cross-links.
 41. The methodof claim 39, wherein said crosslinked tropoelastin consists essentiallyof chemically crosslinked tropoelastin having non-naturally occurringchemical cross-links.
 42. The method of claim 41, wherein saidchemically crosslinked tropoelastin having non-naturally occurringchemical cross-links is crosslinked using glutaraldelhyde as thecrosslinking agent.
 43. The method of claim 39, wherein said crosslinkedpolymer consists essentially of crosslinked recombinant tropoelastin.44. The method of claim 39, wherein said crosslinked tropoelastinconsists essentially of enzymatically crosslinked tropoelastin havingnon-naturally occurring enzymatic cross-links.
 45. The method of claim39, wherein said crosslinked tropoelastin consists essentially ofradiatively crosslinked tropoelastin having non-naturally occurringirradiated cross-links.
 46. The method of claim 45, wherein saidcrosslinked tropoelastin consists essentially of radiatively crosslinkedtropoelastin having non-naturally occurring γ-irradiated crosslinks. 47.The method of claim 46, wherein said -y-irradiated cross-links areproduced by free radicals which result in crosslink formation.
 48. Themethod of claim 39, which further includes organizing the crosslinkabletropoelastin monomer prior to the cross-linking step.
 49. The method ofclaim 48, wherein organizing the crosslinkable tropoelastin monomerprior to the cross-linking step comprises coacervation.
 50. The methodof claim 39, wherein the crosslinkable tropoelastin monomer is combinedwith collagen and then crosslinked to form a crosslinkedtropoelastin-collagen composite.